The present invention generally relates to gels for separation mediums in electrophoretic systems. More particularly, the present invention involves improved separation medium gels and methods for their use in capillary electrophoresis based DNA separations.
For decades electrophoretic separation techniques have been the method of choice for separating charged molecules and in particular for separating proteins. Early electrophoresis applications and many current applications involve applying electric fields across separation medium gels which are prepared in the form of slabs of varying size and thickness. Samples loaded at one end of the slab migrate across the slab under the influence of the electric field. When the sample's charged components have different electrokinetic mobilities they migrate at different rates and physically separate as a result of their differing electrokinetic mobilities. Traditionally, slabs which vary in size from several inches on each side to several feet are fabricated of a separation gel. Typically these gels are crosslinked polyacrylamide or other water swellable gel systems such as agarose or cellulose.
In recent years capillary, electrophoresis (CE) techniques have become the electrophoretic separation method of choice for many biological researchers. Use of capillary electrophoresis for detection of DNA fragments is described by McGregor et al., "Detection of DNA Fragments Separated by Capillary Electrophoresis Based on Their Native Fluorescence Inside a Sheath Flow," Journal of Chromatography A, 680 (1984), 491-496; and Nishiwaka et al., "Separation of Long DNA Fragments by Capillary Gel Electrophoresis With Laser-Induced Fluorescence Detection," Electrophoresis, 1994, 15, 215-220. CE separations involve injecting samples into a buffer filled or gel filled capillary and generating the electric field across the capillary in order to cause sample components to electrophoretically migrate within the capillary. A variety of on-capillary column and off-capillary column detection techniques can be used to detect the components including uv, visible, fluorescence and electrochemical detection. CE offers many advantages over slab gel electrophoresis techniques. CE is available in fully automated systems which include automated injectors and data storage and analysis features and relatively easy to use detection systems. Additionally, CE separations are more rapid than slab gel separations and their separation mediums can be replaced after each analysis for a subsequent analytical run.
Electrophoresis applications have expanded to include a wide range of charged analytes and analytes which can be derivatized to incorporate at least one charge moiety in order to provide the analyte with an electrophoretic mobility. Thus, in addition to proteins, peptides, and amino acids, electrophoretic separation methods are useful for separating derivatized polysaccharides and oligosaccharides, glycoproteins, nucleic acids and oligonucleotides and charged compounds in general. Particularly noteworthy is the demand by The Human Genome project and other large scale DNA sequencing projects for the capability of separating and identifying large numbers of DNA fragments in a single analysis. Because of the huge number of bases which must be sequenced in these projects, the success of the projects largely depends upon the ability to automate and speed the sequencing process. Because electrophoresis is the primary analytical method used for DNA sequencing, rapid DNA sequencing requires electrophoresis techniques which are not only fast but can resolve many bases in a single analysis.
Slab gel electrophoretic methods using slabs fabricated of crosslinked acrylamide can separate and resolve over 600 bases in a single analytical run. A major disadvantage associated with the slab approach is that they require many hours to perform a single analysis.
Capillary electrophoresis, on the other hand can perform a single run and effectively separate up to about 400 bases in less than an hour. Thus, capillary electrophoresis systems incorporating multiple capillaries in a single automated system offer the advantage of being able to analyze multiple samples in less than an hour. The major limitation associated with capillary electrophoresis in DNA sequencing is the separation medium itself. Like slab gel electrophoresis, DNA sequencing samples traditionally have been analyzed using polyacrylamide separation mediums. In order to remove the gel medium from the capillary and replace it with fresh medium the DNA separation mediums used in capillaries preferably have flow characteristics which allow the medium to flow in and out of capillaries. Thus, unlike traditional slab gel separation mediums which typically use crosslinked polyacrylamide having high viscosity and extensive elasticity properties, capillary electrophoresis separation mediums typically include linear polymers or very lightly crosslinked polymer gels which are present at lower concentrations than slab gel separation mediums and which are capable of being pressure forced to flow in and out of capillaries. Many of these separation mediums are based upon polyacrylamide and include denaturants such as urea and/or formamide. The denaturants improve the DNA fragment separation resolution and DNA sequencing read length, but often result in problems connected with their precipitation from the medium. Moreover, urea containing gels have viscosities which are sufficiently high to cause problems in replaceable gel systems and gels incorporating formamide are not stable in aqueous mediums and thus are associated with short shelf lives.
While these denaturing containing polyacrylamide systems work reasonably well there is an ongoing need for separation mediums which provide longer read length and improved resolution. There is also an ongoing need to provide CE separation mediums which incorporate denaturants at sufficiently high concentration without their precipitation from the separation medium. There is further a need to provide CE separation mediums having extended shelf lives and sufficiently low viscosity to allow the separation mediums to easily flow into and out of capillaries.
It is accordingly an object of the present invention to provide separation mediums suitable for use in capillary electrophoresis systems having read lengths of up to over 500 bases. It is also an object of the present invention to provide separation mediums having suitable denaturants which will not precipitate from the medium system, having extended shelf lives and can be used in replaceable gel systems.